Alkaloid derivatives of lysergic acids



Dec. 22, 1964 E. B. CHAIN ETAI.

ALKALoIn DERIvATIvEs oF LYsERGIc ACIDS original'Filed Dec. '7, 1960 oom2 .Ir .mp

fig. 1

United States Patent Otlice 3,162,640 Patented Dec. 22, 1964 3 162,640ALKALOID DERIVA'IVES OF LYSERGIC ACIDS Ernst Boris Chain, Cesare Boninp,and Antonio Tonolo, Rome, italy, assignors to Spciet FarmaceuticiItalia, Milan, Italy, a corporation of Italy Original application Dec.7, 1960, Ser. No. 74,392, now Patent No. 3,060,104, dated Oct. 23, 1962.Divided and ,this application Jan. 19, .1962, Ser- No. 169,989

Claims priority, application Italy July 19, 1960 2 Claims. (Cl.260-2855) This invention relates to new alkaloid derivatives of lysergicacids, which derivatives are useful for conversion to lysergivc acids,and which evidence good pharmacological activity, especially in respectto the D-lysergic acid vderivativ^ This application is a division ofapplication Serial No. 74,392, filed December 7, 1960, now Patent No.3,060,-

The applicants copending US, patent application Serial No. 41,031, tiledJuly 6, 1960, now Patent No. 3,038,840, describes a process for theproduction of alkaloid derivatives of lysergic acid by fermenting, nnderaerobic conditions, an aqneons nutrient medium containing av source ofcarbon, nitrogen and mineral salt with a newstrain of Cplzviceps paspaliStevens and Hall. v

By carrying out the process as described in said application, a mixtureof alkaloids is obtained comprisingprevailingly, D-lysergic acid amideand D-isolysergic acid amide. We have found now that if, after filteringolf the mycelium, the extraction of the culture broth is carried outunder particular conditions of temperature and pH, two new alkaloidderivatives of D-lysergic acid and D-isolysergic acid, having acarbinolamidic structure, are prevailingly obtained.

The process of the invention comprises filtering a culture brothproduced by the fermentation in submerged culture of virnlented strainsof Claviceps paspal Stevens and Hall, whose preparation is described insaid application, from the mycelium, and extracting the broth with anorganic solvent at a pH between 7 and 9 and a temperature between C. and10 C.

The process of the invention is described in greater detail. as follows:

Culture broths containing about 50G-150.0 y/cm of alkaloids (determinedcolorimetrically as ergometrine) which are produced by fermentation, insubmerged culture, of virulented strains of Claviceps paspal'z' Stevensand, Hall, as described in said application, are filtered from themycelium, cooled to a temperature. in the range of 0 C. to 10 C.,preferably 5 C., and the` pH adjusted to Property between 7 and 9,preferably 8, by adding alkali metal carbonates or bicarbonates, such assodium or potassium carbonate or bicarbonate. The extraction of thealkaloids is carried out with an organic solvent cooled in the 5 rangeof 0 to 10 C., such as chloroform or n-butanol 0r a mixture thereof, orether, benzene, ethyl acetate or amyl acetate. Other solvents utilizablehere are simply determined by making solubility tests on the alkaloids.

The alkaloids may then be concentrated and purified by several (i .e.,3-4) successive transfers from the organic solvent to an acidic aqueousphase, such as aqueous 0.1 M tartaric acid, and vice versa, the pH ofthe aqueous phase being adjusted before the extraction with the organicsolvent between 7 and 9 and the temperature always between 0 and 10 C.

The new alkaloid derivative of D-lysergic acid is obtained bycrystallization from the organic solvent, whilst the isomeric derivativeof D-.isolysergic acid is isolated in small amounts from the motherliquor. The new alkaloids may also be isolated by the formation of theirmaleates in ether solution followed by precipitation and fractionalcrystallization, or by an equivalent puriiication process.

It is an advantage of the process of the invention that the derivativeof D-lysergic acid is prevailingly obtained, at the expense of thederivative of D-isolysergic acid, which is known to be of lesspharmacological interest than the corresponding isomer of D-lysergicacid.

The two. new alkaloids o f the invention have the empiric al formulaC18H21O2N3 and when they are reacted with maleic acid in ether thecorresponding maleates, having the empirical formula C2-H25O6N3, are`obtained.

Acidic or alkaline hydrolysisof the new alkaloids of the inventionyields acetaldehydje, which may be determined both qualitatively andquantitatively by the formation of a dinitrophenyl-hydrazone. TheD-lysergic acid amide or B-isolysergic acid amide as the case nia-y be,which may be isolated from the reaction rni-Xture, may be estimated byan alkaline hydrolysis according to Smith and Tim- I nis, I'. Chem, Soc.1936, 1440, which produces D-lysergic acid or D-isolysergic acid, as thecase may be.

The new alkaloid derivatives of D-lysergic acid may be obtained from theisomeric derivatives of D-isol-ysergic acid and, vice versa, by theisomcrizing processes similar to those described in the literature,viz., Smith and Timmis, I. Chem. Soc. 19,36, 1166-.

Ther U.V. spectra are identical to those of other derivativesA ofD-lysergic acid` and D-isolysergieacid (im, at 2-42 mn and 3-12- ma),

The following table lists other physical-chemical properties ofthe twonew alkaloids.

TABLE Derivative of D-lysergic acid Derivative of D-isolysergc acidMelting point Melting point oi maleate, als" (1%, in dinietlylformamide)i y l K Solubl 1359.0. (decomposition beginning).

C; decom osition-be innn C. (dec.) p g l g) chloroforrn, acetone, ethylacetato ingly soluble in cool chloroform, dimethylformamide. sparinglyether, benzene, water. Insolusoluble inether, benzene, water. ble 1npetroleum ether. Rl-(Butauolacetic acidi-water 4:1:5) C64-0.70 0273-077.RE. =R1` new alkaloid/Rf. ergomet- 0.97 1.10.

rme. 1^. R. Spectrum:

Evidentmaxlmatinpp. 2.94; 3.03; 5.99; 6.55; 6.88; 8.85; 3.05;3.41;.6.02; 6.52; 6.89, 7.27; 7.44; n I 12.76; 13.31. 7; 2;'791; 9. 2;12.42; 12.81; 13.32. Bess evident maxlmalulin) 3.29; 3.112;` 3.52; 3.60;7.25; 7.43; 3.28; 3.51; 3.57; 5.81, 8;11, 8.22; 8.58; 7,57; 7.72; 8.10;8.15; 8.58; 9.23; 9.48; 9.60, 10.12; 10.78; 11.56; 11.89; 9.56; 10.02;10.69; 11.46. 14.39. 6,08;7.84;8.31;89 ;13`.55 6.23; 8.77.

Indectlon points (inn) -diurn bicarbonate.

Because `of the .chemical behavior of the two new products of theinventlon, the chemical structure of said compounds iS /H HN CO-NH-CH-CHa VK/Nvf H one alkaloid being a derivative of D-lysergic acidand the other a derivative of D-isolysergic acid.

The new alkaloids, especially the derivative of D-lysergic acid, showgood pharmacological activity as oxytocic substances and are useful asintermediates in the manufacture of D-lysergic acid by the knownprocesses of the literature.

The invention is illustrated by the following examples in which Example1 illustrates the conditions for obtaining the amides of D-lysergic acidand D-isolysergic acid and Example 2 gives the details for obtaining thetwo new alkaloids of the invention. Example 2 is therefore the preferredprocess here.

Example 1.--Small Amounts of the New Alkaloids are Obtained 23 liters ofa fermentation broth, obtained as described in said copendingapplication, and containing 800 'y/ml. of alkaloids (determinedcolorimetrically as ergometrine) are filtered from the mycelium and thepH is adjusted to 8 with a diluted aqueous solution of sodium hydroxide.The alkaloids are extracted at room temperature with a chloroformn-butanol mixture (4:1); from the organic extract the alkaloids areextracted with an aqueous solution of diluted (1: 100) sulphuric acid.The acidic aqueous extract is then made alkaline, to pH 8, and extractedtwice with 1500 m1. of chloroform. The chloroform extract is dried oversodium sulphate and concentrated under vacuum to about 600 ml. Uponcooling 3.07 gm. of D-lysergic acid amide are obtained (M.P. 240 C.after recrystallization from aqueous acetone).

The mother liquor is further concentrated and the residue treated withpetroleum ether. A precipitate forms, is filtered, dried and extractedwith chloroform. D-isolysergic amide is obtained (M.P. 1Z0-130 C. withdecomposition, after recrystallization from methanol). The part which isinsoluble in chloroform comprises a mixture of D-lysergic acid amide andsmall amounts of the two new alkaloids of the invention, which may beisolated as pure products with great diiiculty by fractionalcrystallization or other industrially applicable methods.

Example 2.-Fr Obtaining the New Alkaloids 460 ml. of a filtered (toremove mycelium) culture broth, obtained according to the processdescribed in said copending application and containing 450 'y/cm ofalkaloids (determined colorimetrically as ergometrine), are cooled to 5C. and the pH is adjusted to 8 by adding so- The alkaloids are extractedwith chloroform cooled to 2 to 5 C. (about 500 ml.) and the chloroformextract is extracted with 50 ml. of aqueous 0.1 M tartaric acid.Severaltransfers (3 4) are carried out between the organic solvent andthe dilute tartaric acid and, by operating in the temperature range of 0to C. and by adjusting the pH of the aqueous phase before the extractionwith organic solvent between 7 and 9, a iinal chloroform extract ofabout 20 ml. is obtained.

Example 3.-Degradation of New Alkaloid Derivative of D-Lysergic Acid toD-Lysergc Acid Amide 96 mg. of alkaloid derivative of D-lysergic acid,dissolved in 1.5 ml. of water and 1.5 ml. of ethanol in the fractionalcrystallization.

presence of catalytic amounts of alkali or acid, are heated for one hourunder nitrogen stream. The outgoing gas is passed through a 0.2%solution of dinitrophenylhydrazine in 5 N HC1: from said solution 33 mg.of acetic aldehyde dinitrophenylhydrazone are obtained.

By cooling the reaction mixture, 42 mg. of D-lysergic acid amide areobtained.

In the same manner, D-isolysergic acid amide is obtained from the newalkaloid derivative of D-isolysergic acid.

Example 4.-Degradation of New Alkaloid Derivative of D-Lysergc Acid toD-Lysergc Acid 236 mg. of alkaloid derivative of D-lysergic acid areheated in methanolic l N KOH for one hour. Ammonia is evolved from thereaction mixture. The solution is acidied with 4 N acetic acid: 121 mg.of D-lysergic acid are obtained.

In the same manner the reaction may be carried out on D-lysergic acidamide.

From alkaloid derivative of D-isolysergic acid, D-isolysergic acid isobtained.

By operating at 0 C., 18 mg. of the D-lysergic acid derivative of theinvention are obtained: M.P.=135 C. with decomposition; [a]D22=-{29i2(1% in dimethylformamide); max at 242 mp. and 312 mp.; LR. spectrum asillustrated in FIG. 1 of the accompanying drawing; empirical formulaClaHmOzNa. From the mother liquor, by concentration under vacuum,filtration of the precipitate and succeeding fractional crystallization,small amounts of the D-isolysergic acid derivative of the invention areobtained: M.P.=70 C.; max at 242 ma and 312 mp; LR. spectrum asillustrated in FIG. 2 of the accompanying drawings; empirical formulaC18H21O2N3.

The new two alkaloids, dissolved in methanol react with maleic acid inether, to give the corresponding maleates having the empirical formulaC22H25O6N3.

The two new alkaloids may be obtained also through the formation oftheir maleates, by treatment with maleic acid in ether solution followedby fractional crystallization.

Each of the two new compounds may be transformed into an equilibriummixture of the two isomeric forms, from which both the forms may beisolated, by treatment under acidic conditons, for instance in 10%aqueous acetic acid at C. for one hour, rendering alkaline at loWtemperature, extraction with chloroform and nally For example, in thisWay the total yield of the new alkaloid of D-lysergic acid can beincreased, by conversion of the alkaloid of D-isolysergic acid into thealkaloid of D-lysergic acid.

Either or both the new alkaloids can be degraded into D-lysergic acid orD-isolysergic acid, respectively, by methods known to the art in respectto other amides of said acids, by hydrolysis under alkaline or acidconditions. This can be used to increase the total yield of either ofsaid acids, if desired.

In order that the instant disclosure be complete in itself, weincorporate below the fermentation processes described in our copendingapplication Serial No. 41,031.

The following relates to a process for the production of alkaloidderivatives of lysergic acid, by submerged fermentation with new strainsof Claviceps paspali Stevens and Hall, from which derivatives purelysergic acid can be obtained in known manner.

Nowadays the alkaloid derivatives of lysergic acid are generallyobtained from ergot, that is from natural sclerotia of Clavicepspurpurea (Fr.) Tul. A. The investigators Stoll. et al. (U.S.P.2,809,920) have recently reported the production of such alkaloids bysaprophytic surface culture of a suitable strain of Claviceps purpurea(Fr) Tul. isolated from rye. Others (Abe et al.: I. Agric. Chem. Soc.Japan 25, 1952, p. 458; Taber et al: Canad. J. of Microbiology 4, 1958,p. 611) have described processes for the preparation of alkaloids bysaprophytic surface culture of some particular strains of Claviceps.However such alkaloids do not contain lysergic acid in their moleculeand are different from those obtained by natural sclerotia of Clzvz'cepspurpurea (Fr.) Tul.

In all these investigations, carried out over many years, the productionof the alkaloids occurs only by saprophytic surface culture after -40days of incubation and besides the unitary production is so low as to beimpractical.

Mare recently., Srrusfm at .aL (Australian P- 34.313/58) have. stendhala grasses .fsf the biosynthetic production 0f @ravi allsads .by thecultivation .0f Clavirs purpurea Tul., under essentially anaerobicconditions and with a substantialrednction of cell respiration. Suchconditions present a number of evident disadvantages.

The process of the present invention facilitates the production oflysergic acid derivatives alkaloids in high yields, through a submergedculture of new strains of Claviceps, under aerobic'conditions andstirring, without causing a reduction of the cell respiration. Thissignifies 'that the formation of alkaloids of lysergic acid can becarried out by an industrial fermentation.

lThe organisms employed for the process of the present invention are newstrains of Clavceps paspali Stevens and Hall. It had been found that thestrains Clqviceps paspali Stevens and Hall, which do not produce thelysergic acid derivatives alkaloids by submerged culture, may bevirulented artiicially, to give new strains of .Claviceps-paspali, whichin turn allow said production.

vThe artificial virulentation occurred as follows. Strain F. 97 wasisolated from sclerotia grown on plants of Pasprzlum dzslcum, collectedat Tivoli (Rome) and identitled and classified as ,Claviceps paspalStevens and Hall. Embryos of Rosen 4 n rye were inoculated, beforegermination, with the strain F. 97 and then cultivated in vitro. The newvirulented subspecies were isolated from sclerotia obtained on saidembryos.

The strains which are used in the process of the present invention andare described as new strains of Clavz'ceps paspali Stevens and Hall inthis specification and the claims thereof have been tiled at theIstituto Superiore di Sanita, Viale Regina Elena 299, Rome (Italy), anddenominated by the marks: F-l40; v1**513/1; F-237; F240. The AmericanType Culture Collection of Washington has assigned to the strains F/s13/1, F/23'7, F/240 and F/l40 of Claviceps pqspqli ATCC numbers 13892,13893, 13894 and 13895, respectively.

The process of the invention is therefore one for the production `ofalkaloid derivatives of lysergic acid which comprises fermenting underaerobic conditions an aqueous nutrient medium containing a source ofcarbon, nitrogen and mineral salt with a new strain of Clavicepspaspalz' Stevens and Hall as hereinbefore defined.

The above-said strains have the following morphological characteristics:the colonies, obtained in agar glucosepotato on Petri dishes, have adiameter of 1.5-3 cm. after 10-15 days of cultivationat 27 C.; they areround, having a continuous border and smooth surface, showing awhite-grey aerial mycelium and a brown or dark vegetative mycelium. Theaerial mycelium, velvety and somewhat fasciculated, is constituted byeither simple or Synnematic hyphae, which have a diameter of 3-4p andsepta at a distance of 2.050,u, containing droplets of fat. Thevegetative mycelium is a mat of compact hyphae which have changed theiroriginal structure in a pseudoparenchyma With selerotal structure. Infact the cells have a polygonal form with a diameter of 3-4 X lil-15u,being tightly bound and showing a great number of droplets of fatmaterial. The presence of conidia or clamydospores has never beenobserved. Sporulation has never been obtained even by changing thesources of carbon or nitrogen in the media. Y i

If the colony surface scratched by a needle the Vegetative mycelium,lies under the aerial mycelium, Diesem@ ink 91." iShY 90.191 Theabove-Said characteristicswr'epresent a particular feature of thesestrains, that have never been observed in other strains of Clavicepsisolated.

In submerged culture the mycelium forms groups of small round orirregular pellets, having sizes of 0.1-1 x 0.5-3 cm., somewhat loose,which are constituted by synnemata formed by tightly bound hyphae. Thehyphae have a diameter .of 3`5p and are straight with Very few `lateralbranchings. rThe hyphae contain a great number of droplets of fat, evenat the early stages. The mycelium, in submerged culture, may have ayellow, brown, greygreen or green color, according to the different medaand to the age.

As regards the production of alkaloid derivatives of lysergie acid, thepresent invention is not limited to the use of the described strains,but comprises also the mutants thereof, which may be obtained, efg., bymeans of either a selection or a mutation by the action of UN'. rays orRoentgen rays or'any other mutagenous substance or, particularly, byartilicial infection of either embryos or grasses cultivated in vitro orplants of grasses cultivated both in vivo or in vitro and the saidmutants are to be included in the definition of a new strain of.Clayccps paspalz' Stevens and Hall.

According to our invention the process is preferably carried out bycultivating the above-described organisms, in aerobic conditions and insubmerged culture, both in laboratory flasks and in industrialfermentors, in an aqueous nutrient solution which contains: inorganicsalts, nitrogen sources and carbohydrates or their suitable compoundsacting as carbon sources, until a high yield of alkaloids is obtained.

As regards the inorganic salts, they may be chlorides and/ or nitratesand/ or carbonatos and/or sulphates and/ or phosphates of alkalinemetals, earth alkaline metals, magnesium, iron, zinc and manganese butpreferably MgSO4 and KH2PO4.

The behavior of the strains described in the present invention whengrown in presence of Fe++ and Zn++ in the medium, is different from thatof the strains of Claviceps purpurea described by Stoll et al. (U.S.P.2,809,920). These two elements may decrease the production of alkaloidsmarkedly.

The nitrogen sources may be ammonium salts such as citrate, tartrate,malate, succinate, oxalate, acetate and the like; amino acids and theirmixtures, peptides 0r proteins, their hydrolysates, meat extracts,hydrosoluble fractions of cereal-like corn or wheat; corn malt extract,cornsteep liquor, soya-bean meal, peanut meal, chick-pea meal, cottonbean meal.

The carbohydrates may be glucose, sucrose, starch, dextrins, sorbitol,mannitol, lactose and the like.

The cultivation can be accomplished under aerobic conditions, in surfaceculture or preferably in submerged culture; it may be carried out eitherin laboratory flasks or in fermentors, under stirring or stillconditions and maintaining aerobiosis with air or oxygen. Thefermentation is carried out at a temperature from 22 to 30 C.,preferably at 27 C. and at pH range from 4.2 to 6, preferably at 5.2.The production of the alkaloids generally starts after two days ofculture, reaching the optimum after 7-9 days.

The evaluation of alkaloids content may be effected on the basis ofcolor tests by the Van Urk reaction (Pharm. Weekbled 66, 1929, p. 473)after extraction as follows: the culture broth is alkalinized to pH 8and extracted rst with chloroform and then re-extracted with the aqueousacidic solution (e.g., 1% H2504 or 2% tartaric acid) which is used forthe colorimetric analysis of alkaloids.

The usual procedures of extraction with suitable organic solvents, suchas benzene, chloroform, methylene chloride and the like, or absorptionwith the known absorbent means, such as charcoal, bentonite and thelike, under alkaline conditions, may be used for the separation andisolation of the mixture orthe obtained alkaloids. The mixture, in whichlysergic acid amide and isolysergic acid amide are'p'revalently present,can then be hydrolyzed "7 a with alkali, in known manner, to lysergicand isolysergic acid (J. Chem. Society, 1934, p. 674, and 1936, p.1440). The details of the cultivation will be illustrated by thefollowing examples.

Example 1 The process is carried out in 500 ml. flasks containing 100ml. of a suitable nutrient medium. The flasks are shaken by a rotaryshaker (200 revolutions/minute; eccentric throw: 10 cm.). The optimalincubation temperature is at 27 C. The relative moisture is 23S-90%. hecultivation is carried out in the dark. A flask is inoculated with themycelium which is obtained from a 10 days culture in potato-glucose-agarof one of the above-described new strains of Claviceps paspal Stevensand Hall. The nutrient medium is the following:

Percent Mannitol Succinic acid 1 KH2PO4 0.1 MgSO4-7H2O 0.03 Chick-peameal 0.1

Percent Mannitol 5 Succinic acid 3 KHZPO4 0.1 MgSOt-7H2O 0.03 Distilledwater.

The pH is adjusted to 5.2 with aqueous ammonia solution.

In this medium, `the average production of alkaloids reaches 1000,ag/ml. after 7-9 days incubation.

Ten liters of culture broth, obtained by 110 fermentating flasks, arefiltered and the mycelium is discarded since it contains a very lowamount of alkaloids. The filtered dark colored broth (which containsabout 1000 pgJrnl. of alkaloids) is made alkaline by adding sodiumcarbonate or sodium hydroxide solution and extracted with 10 lt. of amixture chloroform-isobutanol (4:1). The organic extract is re-extractedwith an aqueous 2% tartaric acid solution. The aqueous -acidic solutionis then concentrated under vacuum and at -40 C. to a small volume (aboutone-tenth of the original volume). The residual solution is madealkaline, extracted with chloroform and the solvent evaporated. A whitecrystalline powder is obtained, from which, by alkaline hydrolysis inknown manner, lysergic and isolysergic acid are obtained.

Example 2 The cultivation is carried out with the following nutritivemedium:

The pH is adjusted to 5.2 with aqueous ammonia solution.

The fermentation is carried out according to the procedure described inExample 1. After 7-9 days incubation, the production of alkaloidsreaches the value of 1000 pg/ ml. The same yield is obtained if tartaricacid, citric acid, malic acid, acetic acid, fumarie acid, succinic acidare used.

Example 3 The cultivation is carried out with the following nutrientmedium:

Percent Sorbitol 5 Malic acid 3 KH2PO4 0.1 MgSO4-7H2O 0.03 Distilledwater.

The pH is adjusted to 5.2 with aqueous ammonia solution.

The fermentation is carried out according to the procedure described inExample 1. After 7-9 days incubation, the production of alkaloidsreaches 1000 [lg/ml.

Example 4 The cultivation is carried out with the following nutrientmedium:

Percent Mannitol 4 Glucose 1 Succinic acid 2 KH2PO4 0.1 MgSO4'7H2OChick-pea meal 0.5

Distilled water.

The fermentation is carried out in glass fermentors having a ratio lz/Dnot less than 3. Four liters of the following nutrient medium are pouredinto each fermentor.

Percent Mannitol S Succinic acid 3 KHZPO.; 0.1 MgSO4-7H2O 0.03 Distilledwater.

The pH is adjusted to 5.2 with aqueous ammonia solution.

rl`he sterilization is eected in an autoclave for 20 minutes at C. andfor 40 minutes at 120 C. The fermentors are aerated from the bottomthrough a sintered glass sparger. The foam is controlled by adding theusual anti-foaming agents such as Vaseline oil (Vaseline is a registeredtrademark) containing 6% Alkaterge and the like. The incubationtemperature is kept at 27 C.

The inoculum is constituted by 400 ml. of the culture prepared asdescribed in Example 1. After 4-6 days culture, the production ofalkaloids reaches the optimum The pI-I is adjusted to 5.2 with aqueousammonia slution.

The fermentation is carried out as described in Example After 4-7 daysculture the production of alkaloids reaches the optimum reported inExample 4.

Example 7' The fermentation is carried out in stainless steel fermentorswhich are four meters high and have a diameter of 0.2 m. 90 lt. of thefollowing nutrient medium are poured into each fermentor:

Percent Mannitol 5 Succinic acid 1 KH2PO4 0.1 MgSO4-7H2O 0.03 Chick-peameal 0.1 Distilled water.

The pH is adjusted to 5.2 with aqueous solution.

The sterilization is elfected in another suitable container, so that thecultivation liquid does not come into contact with the direct steam. Infact, it has been observed that the smallest traces of iron in themedium cause a decrease of the production of alkaloids. The inoculum isconstituted by 9 lt. of a culture prepared as described in Example 1.The air enters from the bottom through a porous sparger (1 volume air/ 1volume liquid/ 1 minute).

After 6-9 days incubation the same high yields of alkaloids reported inExample 4 are obtained.

Example 8 The fermentation is carried out in stainless steel fermentorscontaining 50 1t. of the following medium:

Percent Mannitol 4 Glucose 1 Succinic acid 2 KHZPO.; 0.1 MgSO4- 7H2O0.03

Distilled water.

2. The derivative of D-isolysergic acid having the form-ula:

HN l C O--NH-CH-CH3 \N on References Cited in the file of this patentStoll et al.: Helv. Chim. Acta, Volume 26, pages 944-965 (1943), pages956-957 relied on.

1. THE DERIVATIVE OF D-LYSERGIC ACID HAVING THE FORMULA: 